Cancer is the second leading cause of death in the United States. In contrast to other human cancers, incidence and death rates of prostate cancer (PrC) have significantly increased in the current decade. More than 70% of PrC patients will face post-treatment recurrence and transition of the disease to an incurable state. It is largely accepted that human tumors are organized hierarchically, and that the top of this hierarchy is occupied by malignant stem cells, which possess unlimited self-renewal and tumor-initiating capacities. According to the most recent concept of carcinogenesis, only specific phenotypic subpopulation(s) of cancer stem cells (CSCs) are responsible for tumor development, and for the production of the entire spectrum of the differentiated progeny that compose a tumor mass, including metastatic and drug resistant cells. CSCs have been isolated from all major human cancer types, including colorectal, pancreatic and prostate cancers. Numerous studies on many cancer types have demonstrated that the tumorigenic cells expressing common CSC markers, in particular CD133 and CD44, are exceptionally resistant to conventional anti-cancer drugs (such as 5-FU, oxaliplatin, irinotecan, docetaxel and others).
Multidrug resistance (MDR) to conventional and novel chemotherapeutic agents represents a formidable challenge for clinical cancer therapy. While MDR is not exclusively a property of CSC, a great deal of evidence shows that MDR is intimately associated with the presence of CSC. CSCs are naturally resistant to chemotherapy due to multiple mechanisms, including their relative quiescence, their profound capacity for DNA repair, their activation of the ATP-binding cassette (ABC) transporters that efflux many standard anticancer agents, and their resistance to apoptosis. The quiescence of CSCs also promotes their resistance to chemotherapy and radiation therapy. Moreover, the majority of standard anti-cancer drugs actually stimulate quiescent CSCs to self-renew and repopulate the tumor with drug resistant cells. CSC also show a number of phenotypic properties that are critical for the tumor phenotype, such as unrestricted cell replication, self-sufficiency and long-term survival. These properties help to explain why many cancer therapies, while killing the bulk of mature tumor cells, often fail, because they do not eradicate CSCs. Current prostate cancer treatments primarily target the bulk neoplastic, fast-growing cancer cells but not the CSCs subpopulation, and this could provide the reason for the limited survival benefits seen with most prostate cancer therapies. A surviving fraction of CSCs makes tumor recurrence almost inevitable following an apparently successful de-bulking by surgical resection and/or radiation and chemotherapy.
The fact that most cancer drugs do not address the CSC subpopulation explains the fact that the anticancer drugs in development have the highest attrition rate as compared to other diseases: only 5% of agents that have anticancer activity in preclinical development make it through to regulatory approval and even then may only have a small benefit. In particular, current anti-cancer drugs in development for prostate cancer have a significantly lower success rate as compared to other cancers. On the other hand, preclinical evaluation of candidate anticancer agents is traditionally based on the use of unselected high-passage commercial cancer cell lines grown as a monolayer culture. However, long-term in vitro maintenance inevitably leads to the accumulation of additional genomic and epigenomic changes, as well as to the selection of dominant cell subpopulations. Indeed, it was recently demonstrated that the most commonly used established cancer cell lines have no or low correlation with the original clinical samples. This suggests that the use of established cell lines for the study of genomic alterations, discovery of clinically relevant molecular targets, and anticancer drug development is questionable, since the use of these cell lines does not account for the complexity and pathophysiology of in vivo tumors. All of the above considerations highlight the crucial role of CSCs in the discovery of clinically relevant molecular targets and creates an urgent need for CSC-targeted drug development, more physiologically and clinically relevant sources of cancer cells, as well as more relevant in vitro and in vivo models.
Recently, Applicants have established patient-derived ultra-low passage prostate cancer cell line with stable retaining of the features of immature, stem-like cells (PPT2 cell line). The previous studies have demonstrated that the CD133hi/CD44hi phenotype of prostate cancer cells showed clear stem cell-related features, including high tumor- and spheroid-initiating capacities, plasticity (ability to produce multiple cell phenotypes), and high resistance to standard drugs. These cells express over-activated developmental pathways and express high levels of several key transcription factors determining embryonic stem cell pluripotency. In addition, the PPT2 cells express many genes related to anti-apoptotic signaling and drug resistance, which make them a good model for CSC-targeted drug development studies.
Recent studies by Weinberg, Lander, and other groups have shown the tremendous plasticity for cancer cells to interconvert between differentiated tumor and cancer stem cell (CSC) phenotypes. Clinical research efforts show that cells with phenotypic-CSC markers are more prevalent after treatment with traditional chemotherapeutic agents, and are more tumorigenic than their differentiated counterparts. CSCs exist in ‘meta-states’ with significant plasticity, so that these cells can differentiate into cells that are tumorgenic and are either chemosensitive or MDR resistant.
This information suggests a need for therapies that address a number of “meta-phenotypic” states, and are multimodal, in order to mitigate MDR-mechanisms arising from both differentiated tumor cell and CSC populations. First-generation taxoid drugs, such as paclitaxel (PX) and docetaxel, do not meet this need. Taxoid drugs stabilize microtubules and inhibit late G2 or M phases of cell cycle, thereby causing the cell death. Although very active clinically, PX and docetaxel have several clinical problems including poor drug solubility, serious dose-limiting toxicities such as myelosuppression, peripheral sensory neuropathy, allergic reactions, and eventual development of drug resistance. A number of these side effects have been associated with the solvents used for dilution of these antineoplastic agents: Cremophor EL for paclitaxel and polysorbate 80 for docetaxel. In addition, reports have linked these solvents to undesirable alterations in PX and docetaxel pharmacokinetic profiles. A major drawback of the first generation taxoids is there ineffectiveness against MDR cells. These drugs are substrates of P-glycoprotein (Pgp), an effective ATP-binding cassette (ABC) transporter, which actively pumps the drugs out of the cells and induces drug resistance. This helps to explain why PX and docetaxel are effective initially against breast, ovary, and lung cancers, but do not show efficacy against colon, pancreatic, melanoma, and renal cancers. For example, human colon carcinoma is inherently multidrug resistant due to the over expression of Pgp. Accordingly, PX does not show any appreciable efficacy against human colon cancer xenografts in mice.
Second-generation taxoids offer an improved solution to the problems of MDR and CSCs. In sharp contrast with PX, a number of second-generation taxoids, such as SBT-1214, show excellent activity (2-3 orders of magnitude more potent than PX) against drug resistant cancer cells, expressing MDR phenotypes. In several studies using colorectal and prostate cancer models, SBT-1214 was shown to effectively kill both CSCs in vitro and in xenograft models. SBT-1214 was also found to possess intrinsic Pgp modulating ability. SBT-1214, exhibited remarkable efficacy against highly several drug resistant (Pgp+) colon tumor xenografts in SCID mice, inducing complete regression in all surviving mice with tumor growth delay>187 days. The observed total suppression of tumor recurrence by SBT-1214 may indicate that this taxoid can kill or regulate CSCs. Thus, we examined the activity of SBT-1214 against colon CSCs from HCT116, HT-29 and DLD-1 cell lines using cancer spheroids in 3D cultures. Administration of 100 nM SBT-1214 to the HCT116, HT-29 and DLD-1 spheroids for 48 h resulted in marked suppression of the growth of the secondary spheroids in all cells. Most importantly, viable cells that survived this treatment regimen significantly lost the ability to form secondary spheroids, which indicates that colon CSC population was critically affected. Also, it was found that the treatment of HCT116, DLD-1 and HT-29 CSCs with SBT-1214 led to the down-regulation of a number of stem cell-related genes and significant inhibition of genes involved in retaining pluripotency. SBT-1214 inhibited the majority of stem cell-related genes in all colon CSCs examined. It is worthy of note that many of these genes are related to self-renewal, regulation of symmetric/asymmetric division and pluripotency. These results provided strong support for the use of this new-generation taxoid, SBT-1214, as the highly potent cytotoxic antitumor agent component of this study against PPT2 cells and tumors.
A further improvement in taxoid drug delivery was the conjugation of taxoids to natural fatty acids (polyunsaturated fatty acids (PUFAs)). This is an attractive strategy mainly because, (a) some PUFAs possess cancer-specific toxicity via signaling pathways overexpressed in various cancers, (b) various cytotoxic drugs and PUFAS often exhibit synergistic effects against various cancer cell lines, (c) PUFAs appear to have protective effects on healthy cells by preventing drug induced apoptosis, (d) conjugation may decrease systemic toxicity by altering the pharmacokinetic properties of the cytotoxic drugs, and (e) PUFAs are FDA-approved food additives. It has been shown that n-3 PUFAs inhibits the production of carcinogenic eicosanoids derived from n-6 PUFAs through various mechanisms. Eicosanoids that are derived from n-3 PUFAs generally exhibit an inhibitory effect on inflammation and tumor growth. Finally, n-3 PUFAs have been shown to inhibit the ERK1/2 pathway which has been implicated in drug resistance. All of these factors may be contributing to the observed synergy between n-3 PUFAs and a variety of cytotoxic agents.
Among naturally occurring n-3 PUFAs, docosahexaenoic acid (DHA) exhibited the highest potency and thus has been studied extensively. It has been shown that DHA is taken up readily and preferentially by tumor cells for use a biochemical precursor and energy source. Not only does this effect produce a preferential tumor targeting effect, but DHA conjugates also show reduced efflux by Pgp. A DHA conjugate of the first-generation taxoid PX was developed (TAXOPREXIN®: Protarga/Luitpold). The DHA conjugate was found to be is voraciously taken up by tumor cells, internalized (probably through strong lipid-lipid interaction of the DHA moiety with cancer cell membrane), and slowly hydrolyzed by esterases in the cancer cell. DHA does not seem to be a good substrate for Pgp, and was found to reduce the efflux of PX.
The conjugation of DHA to first generation taxoids is not, however, an optimum strategy for overcoming MDR in CSCs and other cancer cells. If cancer cells are over expressing Pgp and/or other ABC transporters, PX molecules, even when released slowly, will be caught by the efflux pump(s) and eliminated from the cancer cells.
Because second-generation taxoids like SBT-1214 already possessed intrinsic resistance to Pgp-mediated efflux, the strategy of making conjugates possibly tumor-targeting by exploiting EPR effects of HAS-fatty acid-taxoid complexes and making tumor-selective transcytosis of HSA complex via Gp 60 by conjugating DHA to these taxoids was developed. The result was the next-generation of taxoids, which include taxoid-fatty-acid conjugates, as exemplified by DHA-SBT-1214 and LNA-SBT-1214.
DHA conjugation to SBT-1214 also provides pro-drug properties, rendering the conjugated drug 10-fold less toxic than free SBT-1214. The DHA moiety shields the taxane backbone and prevents tubulin binding. It is not until the conjugate is taken up by the cell, and the DHA moiety is cleaved by intracellular esterases, that the compound is active.
DHA-SBT-1214 was successfully synthesized and evaluated for its anti-tumor activity against both PX-sensitive and PX-resistant human tumor xenografts in SCID mice. DHA-SBT-1214 was found to cause complete regression of both PX resistant and non-resistant tumors.
The efficacy of DHA-SBT-1214 was evaluated against colon, ovarian, pancreatic and NSCL tumor xenografts in mouse models, which exhibited impressive efficacy. However, in these studies, DHA-SBT-1214 was formulated in solutol HS-15 (or polysorbate 80)/ethanol/saline, and the use of an excipient was found to impose well-documented adverse effects, ascribed to the excipient and ethanol, as well as some stability issues at lower concentration of the excipient. Therefore, Applicants have studied the efficacy of the nanoemulsion formulation, developed in a formulation research laboratory. Despite the robust pre-clinical effects seen with DHA-SBT-1214, there are drawbacks to using the current formulation and Applicants are seeking ways to potentially improve the safety, PK, distribution, retention and ease of use in the clinic.
Although clinically active, taxanes have several issues. These include poor drug solubility, serious dose-limiting toxicities such as myelosuppression, peripheral sensory neuropathy, allergic reactions, and eventual development of drug resistance. A number of these side effects have been associated with the solvents used for dilution of these antineoplastic agents: CrEL for paclitaxel and polysorbate 80 for docetaxel. In particular polyoxyethylated castor oil is biologically and pharmacologically active and leaches plasticizers from standard intravenous (i.v.) tubing releasing di(2-ethylhexyl)phthalate (DEHP). Its infusion produces histamine release with consequent well-described hypersensitivity reactions, including anaphylaxis. In early phase I trials 20% to 40% of un-premedicated patients were affected by these reactions. Moreover it has been also associated with hyperlipidemia, abnormal lipoprotein patterns, aggregation of erythrocytes, and prolonged, sometimes irreversible sensory neuropathy which may be associated with demyelination and axonal degeneration. CrEL can also cause neutropenia. In addition, reports have linked these solvents to the alterations in paclitaxel and docetaxel pharmacokinetic profiles. Hypersensitivity reactions can also occur with polysorbate 80, though to a lesser extent than with CrEL. Polysorbate 80 has also been associated with sometimes severe and irreversible sensory and motor neuropathies. Moreover polysorbate 80 can alter membrane fluidity, leading to cumulative fluid retention. This unique docetaxel toxicity may be reduced by prophylactic corticosteroids. Another important point is that CrEL and polysorbate 80 may limit tumor penetration with a negative impact on efficacy. In particular, the formation of large polar micelles of CrEL-paclitaxel in the plasma compartment entraps the drug and can lead to non-linear pharmacokinetics due to decreased drug clearance and decreased volume of distribution. This contributes to a lack of dose-dependent antitumor activity.
Because DHA-SBT-1214 is extremely hydrophobic, it needs to be formulated in polysorbate 80/ethanol/saline or Solutol H-15/ethanol/saline in order to be infused intravenously. As mentioned earlier, vehicles such as Cremophor and polysorbate 80 produce serious side effects and undesirable effects on pharmacokinetics were mentioned previously.
Nanoscale molecules possess a unique property in their use as the vehicle for anticancer drugs, because of the “enhanced permeability and retention (EPR)” effect. Since the accumulation of nanoscale molecules does not require a specific receptor, the EPR effect is passive in nature, but has been demonstrated to be efficacious. Since the nanoemulsion formulation protocol includes phospholipids and fish oil, the use of DHA-SBT-1214 has a clear advantage over SBT-1214 itself for high affinity to the fish oil component and thus high efficiency in encapsulation, achieving high concentration of the drug inside micelles.
The first line therapy for castration-resistant prostate cancer (CRPS) has been docetaxel with prednisone, and cabazitaxel has been approved by FDA in 2010 in place of or in addition to docetaxel treatment. However, CRPS involving CSCs does not exhibit androgen signaling and thus this type of CRPS is not responding to the combination of docetaxel or cabazitaxel with prednisone.
There is a great need for delivery systems that enhance the solubility, MDR resistance properties, and CSC targeting of PUFA-taxoid conjugates such as DHA-SBT-1214, as well as methods of treating prostate cancer.